The obligate intracellular protozoan parasite Toxoplasma gondii is a major cause of abortion in humans and causes opportunistic infections in immunosuppressed patients such as AIDS patients. Toxoplasma co-opts the host cell by secreting effector proteins into the host cell from specialized organelles called rhoptries (secreting ROPs) and dense granules (secreting GRAs). The cytokine interferon gamma (IFN?) induces a variety of parasiticidal mechanisms in most cells of many species and is believed to be critical to control intracellular pathogens, including Toxoplasma. In mice, IFN? upregulates the immunity-related GTPases (IRGs), which can destroy the vacuole Toxoplasma lives in and subsequently the parasite itself. However, humans lack IFN?- inducible IRGs. Indeed, the Toxoplasma secreted virulence factors ROP18 and ROP5, which cooperatively inactivate murine IRGs, play no role in counteracting the human IFN? response. Thus, despite Toxoplasma?s enormous health implications, little is known about the parasite factors that determine pathogenesis in humans. The goal of this proposal is to systematically identify Toxoplasma genes that contribute to its survival in IFN?- stimulated human cells. In our first aim we will perform genome-wide CRISPR/Cas9-mediated loss-of-function screens to identify Toxoplasma genes that determine fitness in gamma interferon-stimulated human fibroblasts and macrophages. We will generate a pool of loss-of-function mutants by transfecting a Toxoplasma strain expressing Cas9 with a library of gRNAs targeting all Toxoplasma protein-coding genes. These mutants will be used to infect unstimulated or IFN?-stimulated human fibroblasts and human macrophages. We will determine which mutants disappear in IFN?-stimulated but not in unstimulated human cells. By repeating this screen multiple times, we will provide the field with a list of Toxoplasma genes important for survival in IFN?-stimulated human cells. In our second aim we will confirm the top hits from the loss-of-function screens in Aim 1 by generating individual knockout parasites and testing if these have a fitness defect specifically in IFN?- stimulated cells. Identification of novel Toxoplasma genes that determine fitness in IFN?-stimulated human fibroblasts and macrophages might eventually lead to the discovery of novel pathways of how human cells detect and destroy Toxoplasma. Furthermore, if we know what Toxoplasma proteins are essential for its survival in IFN?-stimulated human cells we might be able to use this information to develop new therapies against Toxoplasma.